White blood cell (WBC), or leukocyte, analysis is the enumeration, identification and differentiation of the various subpopulations of leukocytes, such as neutrophils, eosinophils, basophils, and monocytes, lymphocytes, myelocytes, metamyelocytes, blast cells, band cells, etc., in normal and pathologic human blood, plasma or serum. Such analysis is a valuable component of diagnostic hematology, useful in the diagnosis of disease, such as leukemia, the monitoring of treatment, such as chemotherapy, the determination of the need for surgery, and other conditions such as hemorrhage, anemia, monitoring bone marrow transplantation, and other disorders involving blood cell production [See, for example, U.S. Pat. No. 5,360,739; H. Shapiro, Practical Flow Cytometry, 3.sup.rd edit., Wiley-Liss, New York (1995); Davis et al (1990) Pathobiol., 58:99-106; and Hoy, (1990) Bailliere's Clin. Haemat., 3:977-988, among others].
Presently, automated leukocyte analysis is conducted in hematology analyzers by utilizing differences in cell morphology or antibody reactivity. The detection of morphological differences between cell populations uses physical measurements including light scatter, direct current (DC) impedance or radio frequency (RF) conductivity of individual cells or even differential absorption or fluorescence of cells stained with certain specific dyes. Leukocyte analysis methods have used reagents or dyes that specifically stain certain leukocytes a different color, and thus enable one to distinguish between different leukocyte populations. Such multiple color dye stains include Romanowski and Wright stains.
Analysis based on antibody reactivity employs specificity of different antibodies to different antigens that may be expressed in different cell types. A measure of such reactivity is obtained through either fluorescence signal from dyes conjugated to the antibody or signature light scatter signal from particles conjugated to the antibody. While a sensitive method, it can be quite costly and complex, requiring careful and long incubation of reagent in the blood sample.
Alternatively, leukocytes may be analyzed based on their different nucleic acid content. DNA content measurements were used clinically for diagnosis and for determination of effects of drugs on tumor cell proliferation kinetics [M. Andreef, Impulscytophotometrie. Berlin, Springer, 1975]. DNA content abnormalities are common in cancer and leukemia [B. Barlogie et. al. Amer. J. Med. 69:195, 1980]. Leukocyte analysis based on nucleic acid content of cells using metachromatic dye stains, i.e., dyes that absorb and/or fluoresce differentially when bound to DNA and RNA nucleic acids, has been suggested. See, e.g., U.S. Pat. No. 4,440,370. As a result, based on DNA or RNA content, different blood cell populations absorb, or fail to absorb, a single metachromatic dye in some unique manner so that each dye-sorbed cell reflects an individual and different light spectra. Such dyes include, without limitation, basic orange 21 (CI#48035), basic red 13, basic red 36, basic red 49, basic violet 7, basic violet 16, among others, and several classes of oxazine dyes described in U.S. Pat. No. 4,400,370, cited above. The process of leukocyte differentiation with metachromatic dyes is quite costly, due to the requirement for at least two photomultiplier tubes (PMTs).
U.S. Pat. No. 4,882,284 describes a method for discriminating WBCs from RBCs and platelets in a whole blood sample by utilizing a metachromatic red light absorbing fluorescent dye, i.e., an oxazine, a red light source and detectors. The dyes described therein include oxazine 170 (also known as oxazine 720). According to this method, the red cells are not lysed. Thus, this method suffers from some disadvantages. Inability to lyse the red cells requires too large of a data collection if the goal is the analysis of leukocytes, due to the 1000:1 ratio of RBC to WBC in normal blood. This aspect of the '284 process makes it undesirable for automated analysis. In another method, U.S. Pat. No. 5,360,739 also describes the use of a unique oxazine dye, oxazine 750, to identify and enumerate reticulocytes. A significant disadvantage of the methods of the '284 and '739 patents is that dyes such as oxazine 720 and oxazine 750, are brightly fluorescent, whether or not they are bound to nucleic acid. In fact, due to their bright fluorescence in free solution, both oxazine 720 and oxazine 750 have been used as gain media in dye lasers. For flow cytometric analysis of cells, however, bright fluorescence of unbound molecules is a highly undesirable attribute for dyes used to stain specific components within the cell. Such fluorescence creates a background noise from non-specifically bound or unbound molecules and diminishes the specificity of detection for the targeted components in the cells.
Certain known dyes such as TOTO-1.RTM. dye, TOTO-3.RTM. dye, TOPRO-3.RTM. dye, etc., and the new SYTO.RTM. dyes, are also capable of staining both DNA and RNA nucleic acid in a cell. SYTO.RTM. dyes are red fluorescent nucleic acid stains in a solvent of dimethylsulfoxide (DMSO), which are commercially available (Molecular Probes, Inc., Eugene, Oreg.). These nucleic acid dyes are sold under the trademarks SYTO.RTM.17, and 59 through 64. These stains have been reportedly employed for detection of live or dead bacteria, e.g., C. parvum viability [M. Belosevic et al, (1997) Int'l. J. Parasitol., 27:787] and in methods for microbiological characterization and viability assessment [P. J. Millard et al, (1997) Biotechnol. Int'l., 1:291]. The manufacturer describes these stains as cell-permeant nucleic acid stains that differ from each other in characteristics including cell permeability, fluorescence enhancement upon binding, and in excitation and emission spectra. Eukaryotic cells incubated with SYTO.RTM. dyes reportedly show cytoplasmic or mitochondrial staining as well as nuclear staining (Molecular Probes product insert). These dyes have also been reported to produce minimal background fluorescence from unbound dye and enhancement of fluorescence quantum yield of DNA and RNA bound dye molecules compared to unbound dye molecules. However, none of these dyes have been suggested as possible agents for WBC or reticulocyte/mature RBC differential analysis.
In view of the importance of blood cell population analyses, e.g., leukocyte differentiation, there exists a continuing need in the art for methods which improve blood cell differentiating technology, such as by enabling rapid staining and differentiation of intracellular nucleic acids and permitting ready and accurate detection of white blood cell (WBC) subpopulations and other blood cell populations, such as RBC and reticulocytes.